Establishment Of Chicken Infectious Anemia Virus After Infection Of Host Gene Expression Profiling And Real-time PCR Detection Method

Chicken infectious anemia virus can cause aplastic anemia and lymphoid tissue atrophy, which is one of the important causes huge economic losses poultry industry.The microarray,which belongs to a gene microarray chips and is one of the most widely used,can be used to detect the host genome and the study of gene expression under different conditions.Real-time PCR is by accumulating PCR products,so that the fluorescent signal intensity increased in proportion to the real-time monitoring of amplification products.This paper studies two parts.The first part is the expression of chicken infectious anemia virus infection in chickens after spectral analysis,and thesecond part is to establishrealtime PCR detection methods. 1. the expression profile of chickens infected with chicken infectious anemia virus analysisChicken infectious anemia mainly infects chickens,through horizontal and vertical transmission,resulting in a large number of dead chickens.To better understand the pathogenic mechanisms,we first set upanimal model experiment.CIAV infect1-day-old SPF chickens,while we have the establishment of a negative control.We observe at the onset of the worst 14 day-old chicks,the most obvious symptom.We take the chicken thymus extract total RNA,reverse transcribed into c DNA,and then synthesize c RNA.We using Affymetrix whole genome expression arrays and their hybridization,to sudy the changes in gene expression profiling.Our data on the differences in gene expression were obtained by GO analysis and Pathway analysis.We use DAVID software to analyze its biological function,and apply of real-time PCR toverify microarray results.The results were screened 2290 differentially expressed genes, including 1760 up-regulated genes and 530 down-regulated genes,Which include 38 immune-related genes.We use SAM software to filterthe differentially expressed genes,and use cluster to analysis gene expression differences.By using DAVID software,we get the biological function of the gene expression analysis.Meanwhile,We use the real-time PCR to verify the differences among the seven genes,which the results with the microarray results are basically the same, indicating that microarray reliable. 2., The establishment of real-time PCR detection methodReal-time PCR for sample testing is accurate,sensitive,and can accurately quantify in sample testing.It makes up for virus isolation and identification,lack of serological diagnosticdetection methods,PCR diagnostic methods,electron microscopy and measurement of hematocrit values,etc.Real-time PCR includes probe and dye method.The SYBR Green? dye is operated simply,lowcost,therefore it is widely used.The experiment was based on chicken infectious anemia virus genome conserved regions to designe for 180 bp amplified fragment size specific primers.We build PGM-T-CIAV recombinant plasmid preparation positive standard, and establish SYBR Green? quantitative PCR standard curve.We do the sensitivity test,specificity test and reproducibility tests,and clinical samples were tested at the same time.Real-time PCR experiment,compared with conventional PCR,has high sensitivity and specificity, repeatability.And in the detection of c l i n i c a l s a m p l e s, i t i s p r o v e d i t s h i g h s e n s i t i v i t y o n c e again.